Murine leukemia virus preferentially integrates within active promoters regions, proximal to transcriptional start sites (TSS) and CpG islands. The site of integration is linked to the oncogenic potential of the virus and thus its pathogenesis and use as a gene delivery vector. Our collaborative research has recently identified the host BET family of proteins, specifically the Brd 2, 3, and 4 proteins as interactors with the MLV IN and this interaction influences the integration into TSS and CpG islands. This provides the first window into our understanding of the mechanism of MLV target-site selection. This application studies the newly defined viral-host interaction between the MLV IN and BET family members. Four specific aims are built on two key observations. The first links the binding of Brd ET domains to the MLV IN C-terminal domain. The second indicates that IN proteins lacking the C-terminal 23 amino acids are no longer biased towards integration to TSS and CpG islands. Based on the solution structure of the MLV IN CTD from our laboratory, the first specific aims at defining the NMR structure of the MLV IN CTD:Brd3 ET complex. Additional structural analysis of the CTD in complex with the crossbone DNA integration intermediate will be pursued. The second specific aim applies mutational and biochemical assays to define the primary as well as potential secondary Brd binding sites. The controversial role of the IN CCD in binding BET proteins will be examined. The ability of the MLV IN protein to compete with known ET domain interactors will be studied. The third specific aim utilizes next-generation sequencing and bioinformatics to probe a panel of IN proteins for their target-site preferences, both locally at the site of integraion as well as globally within the human genome. The recognition of the target DNA within the nucleosome structure is examined. The final specific aim applies this information towards developing targeted integrating vectors. The research translates our strong knowledge of MLV IN towards rapidly understanding this interaction with the host target cell. The research addresses one of the outstanding questions relating to the mechanism of targeting MLV integration into promoter regions. These studies have the potential to be applicable to multiple gammaretroviruses. The goal of the research is to apply this knowledge towards decreasing the effects of promoter/enhancer insertions on oncogene activation in gene delivery systems.